1. Field of the Invention
The present invention relates to the fields of oncology, genetics and molecular biology. More particular the invention relates to the identification of the gene responsible for familial juvenile polyposis. Defects in this gene are associated with a predisposition to gastrointestinal cancers.
2. Description of Related Art
Colorectal cancer is the second leading cause of cancer death in the United States. and was responsible for 57,407 deaths in 1994 (Landis et al., 1998). Approximately 5-10% of the nearly 131,600 new colorectal cancer cases each year will involve a clear heritable predisposition, of which the majority of cases involve hereditary non-polyposis colorectal cancer (HNPCC). About 1% of new colorectal cancers are related to inherited polyposis syndromes, which include familial adenomatous polyposis (FAP) and familial juvenile polyposis (FJP) (Rustgi, 1994).
Identification of the genes responsible for HNPCC and FAP have greatly increased our understanding of the molecular mechanisms contributing to the development of both familial and sporadic colorectal cancer. The intense studs of sporadic colorectal carcinogenesis over the last decade has shown that these tumors develop through the multi-step accumulation of different genetic mutations within colonic epithelial cells (Vogelstein et al., 1988). Genes known to be involved in this progression include APC and MCC on 5q21, KRAS2 on 12p12, p53 at 17p13, and several mismatch repair genes as seen in HNPCC (reviewed in Howe and Guillem, 1997).
Deletions on 18q21 also are quite common, occurring in approximately 75% of colorectal cancers (Vogelstein et al, 1988). Initial studies suggested that the tumor suppressor gene DCC (deleted in colorectal cancer)was the predisposing gene from this region (Fearon et al., 1990), but this has not been clearly established by further investigation.
Familial juvenile polyposis (JP) is an autosomal dominant condition characterized by multiple juvenile polyps of the gastrointestinal (GI) tract. Kindreds have been described in which there is involvement of the colon only juvenile polyposis coli, MIM 174900) (Veale et al., 1966; Grotsky et al, 1982; Rozen and Baratz 1982), the upper GI tract (Watanabe et al., 1979), and both upper and lower GI tracts (generalized polyposis) (Sachatello et al., 1970; Stemper et al., 1975; Jarvinen and Franssila 1984), although whether these are distinct clinical entities is not clear. Affected family members often present with blood per rectum or anemia in the 2d decade of life (Jass et al., 1988).
Microscopically, the polyps contain cystically dilated glands, abundant stroma. and an inflammatory infiltrate (Morson 1962). There have been many reports of patients with juvenile polyposis developing gastrointestinal malignancy, including colon cancer (Stemper et al, 1975; Liu et al., 1978; Goodman et al. 1979; Rozen and Baratz 1982;
Jarvinen and Franssila 1984; Ramaswamy et al, 1984; Baptist and Sabatini 1985; Jones et al, 1987; Bentley et al, 1989; Scott-Conner et al, 1995), stomach cancer (Stemper et al, 1975; Yoshida et al, 1988; Scott-Conner et al. 1995), and pancreatic cancer (Stemper et al., 1975; Walpole and Cullity 1989). Affected family members"" risk of developing GI malignancy has been estimated to be from 9% (Jarvinen and Franssila 1984) to as high as 50% (Jass 1990). Development of adenocarcinoma has been hypothesized to begin with an adenomatous focus within a juvenile polyp, which later becomes dysplastic, and finally undergoes malignant transformation (Goodman et al., 1979; Jarvinen and Franssila 1984).
JP is a hamartomatous polyposis syndrome, as are Peutz-Jegher""s Syndrome (PJS) and Cowden""s disease (CD). Although the polyps in PJS are true hamartomata, some may undergo adenomatous change, and these family members are at increased risk for gastrointestinal malignancy. The PJS gene was mapped to chromosome 19p by comparative genomic hybridization and linkage (Hemminki et al., 1997; Mehenni et al., 1997), and germline mutations were identified in the serine threonine kinase gene LKBI (Hemminki et al., 1998). In CD, affected family members may develop multiple hamartomata of the skin, breast, thyroid, oral mucosa, or GI tract, and they are at risk for breast and thyroid malignancies. The gene for CD was localized to chromosome 10q22-23 by linkage (Nelen et al, 1996), and germline mutations in the PTEN gene have been found in affected family members (Liaw et al., 1997). A third entity, termed the xe2x80x9chereditary mixed-polyposis syndromexe2x80x9d (HMPS), differs from these syndromes in that affected family members have atypical juvenile polyps, colonic adenomas, and colorectal carcinomas. A gene for HMPS has been mapped to chromosome 6q by linkage (Thomas et al., 1996), and it remains uncertain whether HMPS is a distinct clinical syndrome or a variant of FJP (Whitelaw et al., 1997).
To date linkage studies in JP families have been limited, with one report excluding APC and MCC as the genes for FJP (Leggett et al., 1993). Other genetic studies, originally stimulated by the finding of an interstitial deletion at 10q22-24 in an infant with multiple colonic juvenile polyps and several congenital abnormalities (Jacoby et at., 1997b), have focused on the region of the PTEN gene. Evaluation for loss of heterozygosity in this region within juvenile polyps revealed somatic deletions within the lamina propria in 39 (83%) of 47 polyps derived from 13 unrelated patients with familial JP and 3 patients with sporadic juvenile polyps. These findings have been interpreted as evidence for a tumor-suppressor gene on 10q for FJP (termed xe2x80x9cJPIxe2x80x9d) (Jacoby et al., 1997a), but a recent study of 14 FJP families found neither mutations in PTEN nor evidence of linkage to markers on 10q22-24 (Marsh et al, 1997). Analysis of an additional 11 cases of FJP also did not uncover mutations in the PTEN gene (Riggins et al., 1997). Lynch et al., (1977) reported one family thought to have both juvenile polyposis syndrome and CD as having a nonsense mutation in PTEN, and Olschwang et al., (1998) described three patients with juvenile polyposis as having PTEN mutations. Whether these four individuals should truly be considered as having juvenile polyposis rather than CD is not clear from these reports.
It is evident from the discussion presented above that FJP is a significant disease which has yet to be definitively linked to aberrations in a particular gene. The identification of such a gene will allow the determination the molecular basis of gastrointestinal polyposis predisposing to colorectal cancer, as well as presymptomatic diagnosis of family members at risk. Such a gene also may be involved in the genesis of sporadic colorectal cancers, and therefore its discovery could ultimately impact on the treatment of this large group of patients.
A particular objective of the present invention is to identify the JP gene in a large kindred with generalized juvenile polyposis and gastrointestinal cancer and to use the gene in various diagnostic and therapeutic applications.
Thus in a preferred embodiment, the present invention provides a method of diagnosing juvenile polyposis comprising the steps of obtaining a sample from a subject; and determining the loss or alteration of a functional SM4D4 gene in cells of the sample. In certain defined embodiments the sample may be selected from the group consisting of blood, buccal smear and amniocentesis sample. In still further embodiments, the sample may be a tissue or fluid sample. In preferred embodiments, the determining may comprise assaying for a nucleic acid from the sample. In still further embodiments, the determining may further comprise subjecting the sample to conditions suitable to amplify the nucleic acid.
In alternative preferred embodiments, the determining comprises contacting the sample with an antibody that binds immunologically to a SMAD4. In particularly preferred embodiments, the method further comprises subjecting proteins of the sample to ELISA. In particular aspects of the present invention, the method may comprise the step of comparing the expression of SMAD4 in the sample with the expression of SMAD4 in non-juvenile polyposis samples. In defined aspects of the invention, the comparison involves evaluating the level of SMAD4 expression. In other aspects the comparison involves evaluating the structure of the SMAD4 gene, protein or transcript. In more defined embodiments, the evaluating may comprise an assay selected from the group consisting of sequencing, wild-type oligonucleotide hybridization, mutant oligonucleotide hybridization, SSCP, PCR(trademark), denaturing gradient gel electrophoresis and RNase protection. In other defined embodiments, the evaluating is wild-type or mutant oligonucleotide hybridization and the oligonucleotide is configured in an array on a chip or wafer.
In particularly defined embodiments, the juvenile polyposis sample comprises a mutation in the coding sequence of SMAD4. In other defined embodiments, the mutation produces a deletion mutant, an insertion mutant, a frameshift mutant, a nonsense mutant, a missense mutant or splice mutant. In particularly preferred embodiments, the mutation is a frameshift mutation. In still further defined embodiments, the mutation results in a premature termination of the SMAD4 gene product. In particularly preferred embodiments, the mutation may independently be in exon 9, exon 8, exon 5 or in a combination of exons of the DPC4 gene. In certain preferred embodiments, there is a frameshift that results from a deletion in codons 414 through to 416 of SMAD4. In other defined embodiments, the frameshift results in a STOP at codon 434 of wild-type SMAD4. In yet another alternative, the frameshift results from a deletion in codon 348. In still another alternative the frameshift results in a STOP at codon 350 of wild-type SMAD4. In further embodiments, the frameshift results from a deletion in codon 345. In yet another alternative, the frameshift results in a STOP at codon 382 to 383 of wild-type SMAD4. In another alternative, the frameshift results from an insertion in codon 229 through to 231. In defined embodiments, the frameshift may result in a STOP at codon 235 of wild-type SMAD4. In other embodiments, the mutation is a missense mutation, wherein the mutation is an A to C substitution at codon 352, converting a tyrosine to a serine. In other embodiments, the mutation is a nonsense mutation, wherein the mutation is a G to C substitution at codon 177, converting a serine to a stop codon.
Also contemplated by the present invention is a method for altering the phenotype of a cell in a subject having juvenile polyposis comprising the step of contacting the cell with SMAD4 under conditions permitting the uptake of the SMAD4 by the cell. In particularly preferred embodiments, the cell is derived from a gastrointestinal cell. In more particular embodiments, the phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth and cell cycling. In particularly defined aspects of the invention, the SMAD4 may be encapsulated in a liposome.
In yet another aspect of the present invention there is provided a method for altering the phenotype of a cell in a subject having juvenile polyposis comprising the step of contacting the cell with a nucleic acid (i) encoding SMAD4 and (ii) a promoter active in the cell, wherein the promoter is operably linked to the region encoding the SMAD4, under conditions permitting the uptake of the nucleic acid by the cell. In particular aspects the cell may be derived from a gastrointestinal cell. In other embodiments, the cell is a tumor cell. In certain defined embodiments, the nucleic acid may comprise a viral vector selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, vaccinia virus and herpesvirus. In particular embodiment, the nucleic acid is encapsulated in a viral particle.
Also contemplated is a method for treating juvenile polyposis comprising the step of contacting a cell within a subject with SMAD4 under conditions permitting the uptake of the SMAD4 by the cell. In particular preferred embodiments, the subject is a human.
In yet another embodiment, the present invention contemplates a method for treating juvenile polyposis in a subject comprising the step of contacting a cell within the subject with a nucleic acid (i) encoding SMAD4 and (ii) a promoter active in the cell, wherein the promoter is operably linked to the region encoding the SMAD4, under conditions permitting the uptake of the nucleic acid by the cell. In particular aspects of the present invention the cell may be derived from a tissue selected from the group consisting of skin, muscle, fascia, brain, prostate, breast, endometrium, lung, head and neck, pancreas, small intestine, blood cells, liver, testes, ovaries, colon, rectum, skin, stomach, esophagus, spleen, lymph nodes, bone marrow and kidney.
In other embodiments, it is contemplated that detecting SMAD4 expression levels can be utilized prognostically in identifying colorectal cancer.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 Pedigree of a five-generation American midwestern FJP family (the Iowa FJP kindred). Blackened symbols designate affected family members; unblackened symbols indicate unaffected individuals or those with unknown affection status; genotyping was performed on those marked by asterisks (*).
FIG. 2A and FIG. 2B. Schematic representation of STRPs and genes from 18q21 (FIG. 2A) and recombination analysis of key affected individuals (FIG. 2B). STRPs are listed in their map order, based on the Center for Medical Genetics map; markers D18S977 and D18S849 lie between D18S858 and D18S862 (Whitehead Institute for Biomedical Research/MIT Center for Genome Research); and D18S46 lies between DPC4 and D18S363 (Hahn et al., 1996a). The locations of MADR2, SSAV1, DPC4, and DCC are representations derived from physical mapping data (Eppert et al., 1996); the lengths of the corresponding bars shown for the map at left do not necessarily reflect the size of each gene. To the right, informative recombination events in affected individuals that define the interval of the JP gene are depicted as blackened boxes; unblackened boxes designate noninformative meioses. These data suggest that the JP gene lies between the markers D18S1118 and D18S487.
FIG. 3A and FIG. 3B. Sequences of the wild-type (FIG. 3A) and mutant (FIG. 3B) alleles of SMAD4 exon 9 (nucleotides 1365 to 1382) from an affected member of the Iowa JP family. The rectangle indicates the 4 base-pairs deleted in the mutant allele (arrow).
FIG. 4A and FIG. 4B. Denaturing (FIG. 4A) and nondenaturing gels (FIG. 4B) of Iowa JP kindred family members, showing the SMAD4 exon 9 PCR product. Affected individuals 4, 5, 6, and 11, as well as one at risk (8), all have an extra band on denaturing gels that is produced by the 4 base-pair deletion. The mutant allele is also seen as a shift by SSCP (FIG. 4B).
FIG. 5. PCR amplification of SMAD4 exon 9 from microdissected polyps. Pedigree numbers correspond to affected individuals as described in the Examples. Loss of the wild-type allele is seen in a juvenile polyp from patient IV-17 (arrow). DNA was extracted from paraffin-embedded polyps after microdissection. Amplification of exon 9 was performed using the primers 5xe2x80x2-TAGGCAAAGGTGTGCAGTTG-3xe2x80x2 (SEQ ID NO:1) and 5xe2x80x2-TGCACTTGGGTAGATCTYATGAA-3xe2x80x2 (SEQ ID NO:2), which generate a 152 bp product from within the exon. C-colon; S-stomach; VA-villous adenoma-AP=adenomatous polyp; JP=juvenile polyp.